Epithelial-mesenchymal cell interaction and factors that control normal lung development are also key players in lung injury repair and fibrosis. Many studies have investigated the role and source of epithelial progenitors during regeneration. There is limited information on the origin and fate of mesenchymal progenitor cells, however, even less knowledge about epithelial mesenchymal crosstalk during injury and repair. The long-term goal of this work is to understand the complexity of fibroblast populations and to identify their role in lung generation and regeneration. The objective in this application i to identify key regulators of fibroblast differentiation, identify fibroblasts that promote alveola epithelial cell differentiation and to determine age dependent modifications that regulate differentiation of fibroblasts. Based on strong preliminary data, the central hypothesis is that GATA6 is a key regulator of the matrix gene signature and that loss of matrix fibroblasts disturbs epithelial-mesenchymal interactions. The rationale for the proposed research is that understanding the regulation of fibroblast subpopulations, and their distinct role in epithelial-mesenchymal cell interactions, will provide new answers for fundamental questions regarding lung disease and regeneration. This hypothesis will be tested with three specific aims: 1) determine the function of GATA6 in regulating the matrix gene signature and fibroblast differentiation during alveolar regeneration 2) determine the role of the matrix fibroblast in alveolar epithelial cell differentiation 3) identify the epigenetic modifications that regulate fibroblast differentiation in aged lungs. In the first aim, GATA6 gain and loss of function studies will be used to establish a functional link between alveolar regeneration and differentiation plasticity of interstitial fibroblasts. In the second aim, alveolosphere cultures will be used to interrogate the epithelial-mesenchymal crosstalk between Gata6 mutant interstitial fibroblasts and wild type alveolar epithelial cells. In the third aim, epigenetic Gata6 promoter modifications by EZH2 will be assessed by ChIP-PCR analysis. In vivo and in vitro studies using EZH2 inhibitors will determine a role of EZH2 in suppressing Gata6 and matrix fibroblast signature genes. These studies will provide a better understanding of myo and matrix fibroblast differentiation and the impact of fibroblast diversity on epithelial-mesenchymal crosstalk during realveolarization. This contribution will be significant because it will advance the knowledge about genes and pathways involved in alveolar regeneration, give new insights into the cause of decreased regrowth with age and provide the basis for new therapies to overcome current limitations of regeneration in the human lung. Our understanding of the role of lung fibroblasts in lung injury and regeneration lags far behind our understanding of the role of epithelial cells. New information about interstitial lung fibroblasts will be invaluable for defining mechanisms of normal lung repair and pathologic lung disease development.